Figure 3.

Time-lapse observation of gTOW experiment with the target gene mik1+. (A) FY7652 cells harboring the pTOWsp-H plasmid with the mitotic CDK inhibitor mik1+ were cultivated in EMM without leucine and were observed under fluorescent microscopy. The plasmid copy number in each cell was estimated from the intensity of GFP fluorescence. The black arrowhead with a white frame indicates a cell with very low plasmid copy number to synthesize a sufficient amount of leucine to support growth. The cell stops proliferating after the second cell division (frame 6). The white arrowhead indicates a cell with a sufficiently high plasmid copy number to synthesize enough leucine to support rapid cellular growth, but this number is beyond the copy number limit of mik1+. The cell thus cannot divide, becomes elongated (frames 3–5), and eventually dies (frame 6). The yellow arrowhead indicates cells with balanced plasmid copy number; the cells can grow and divide because they synthesize sufficient leucine. Cells with a plasmid copy number close to the limit of mik1+ thus become concentrated during cultivation. A video clip is provided in Supplementary information. (B) Assumed cellular situations in gTOW experiment are shown (A). The arrowheads correspond to the arrowheads in (A).